Putative biological roles of hydrogen sulfide in health and disease: a breath of not so fresh air?

The past two decades have seen an upsurge in interest in the biology of naturally occurring gases, starting with nitric oxide and extending through to carbon monoxide. The latest addition to the list of biologically relevant gases is hydrogen sulfide. In the past few years, hydrogen sulfide has transited rapidly from environmental pollutant to biologically relevant mediator with potential roles in several physiological processes and disease states. Further, interest is now being shown in developing drugs which either mimic its effects or block its biosynthesis. Similarly to its gaseous cousins, the biology of hydrogen sulfide is proving to be complex and difficult to unravel.

Hydrogen sulphide in the hypothalamus causes an ATP-sensitive K+ channel-dependent decrease in blood pressure in freely moving rats.

Hydrogen sulfide (H2S) is a naturally occurring gas that may act as an endogenous signaling molecule. In the brain, H2S is mainly produced by cystathionine beta-synthase (CBS) and its cellular effects have been attributed to interactions with N-methyl-D-aspartate (NMDA) receptors and cyclic adenosine 3',5'-monophosphate (cAMP). In contrast, direct vasodilator actions of H2S are most probably mediated by opening smooth muscle ATP-sensitive K+ (K(ATP)) channels. In the hypothalamus, K(ATP) channel-dependent mechanisms are involved in CNS-mediated regulation of blood pressure. In this report, we investigated the hypothesis that H2S may act via K(ATP) channels in the hypothalamus to regulate blood pressure. Mean arterial blood pressure (MAP) and heart rate were monitored in freely moving rats via a pressure transducer placed in the femoral artery. Drugs were infused via a cannula placed in the posterior hypothalamus. Infusion of 200 microM sodium hydrogen sulfide (NaHS), an H2S donor, into the hypothalamus of freely moving rats reduced MAP and heart rate. Infusion of 300 nM to 3 microM gliclazide dose-dependently blocked the effect of 200 microM NaHS. Infusion of the CBS activator, s-adenosyl-L-methionine (0.1 mM and 1 mM), likewise decreased MAP. Infusion of the CBS inhibitors aminooxyacetic acid (10 mM) and hydroxylamine (20 mM) increased MAP but did not block the effects of infusion of 200 microM NaHS. These data indicate that actions of H2S in the hypothalamus decrease blood pressure and heart rate in freely moving rats. This effect appears to be mediated by a K(ATP) channel-dependent mechanism and mimicked by endogenous H2S.

A nociceptive-intensity-dependent role for hydrogen sulphide in the formalin model of persistent inflammatory pain.

The present study investigated the hypothesis that hydrogen sulfide (H2S) is pro-nociceptive in the formalin model of persistent inflammatory pain in the adult rat. Hind paw injection of formalin evoked a concentration-dependent increase in the hind paw concentration of H2S. Increased concentration of H2S was found in homogenates prepared from hind paws injected with 5% (but not 1.25%) formalin. Correspondingly, animal nociceptive flinching and hind paw edema were maximal with 5% formalin. Both nociceptive flinching and hind paw edema induced by injection of 5% formalin were attenuated by pretreatment with DL-propargylglycine (PPG; 50 mg/kg, i.p.) which is an inhibitor of the H2S synthesizing enzyme cystathionine-gamma-lyase (CSE). The effect of pretreatment with PPG was selective and the drug did not influence animal behavior or hind-paw edema with injection of 1.25% formalin. Furthermore, PPG pretreatment attenuated the induction of c-Fos in spinal laminae I-II following injection of 5% formalin. In contrast, co-injection of 1.25% formalin with sodium hydrogen sulfide (NaHS; 1 nmol/0.1 ml), a H2S donor, into the hind paw increased animal nociceptive behavior. Collectively, these findings show that the effect of peripheral H2S in the pathogenesis of inflammatory pain depends, at least in part, on the nociceptive intensity level.

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom.

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.

Effect of a nitric oxide releasing derivative of paracetamol in a rat model of endotoxaemia.

BACKGROUND AND PURPOSE: Nitroparacetamol is a nitric oxide-releasing paracetamol with novel anti-inflammatory properties compared to the parent compound. This study has investigated the anti-inflammatory activity of nitroparacetamol in a model of endotoxaemia in rats to probe the mechanisms underlying this effect. EXPERIMENTAL APPROACH: Nitroparacetamol (92 mg kg(-1)), paracetamol (50 mg kg(-1)) or vehicle were administered to male, Wistar rats 15 min prior to or 3 h after lipopolysaccharide (0.5 mg kg(-1), serotype 0127:B8). Mean arterial pressure and heart rate were measured for 5 h and plasma and organs were then obtained to determine organ dysfunction, inducible nitric oxide synthase and cyclooxygenase-2 expression (lung, liver and kidney tissue) and plasma nitrate/nitrite. In separate experiments, nitroparacetamol, paracetamol or vehicle was administered 1 h before acetylcholine (0.1 microg kg(-1)) or sodium nitroprusside (0.25 microg kg(-1)) to determine if nitroparacetamol desensitizes responses to exogenous/endogenous nitric oxide. KEY RESULTS: Nitroparacetamol prevented but did not reverse the lipopolysaccharide-induced hypotension. There was no effect on heart rate or plasma markers of organ dysfunction. Nitroparacetamol prevented the increased plasma nitrate/nitrite and expression of COX-2 and iNOS, whereas paracetamol exerted partial inhibition of COX-2 in lung alone. Nitroparacetamol also reduced responses to acetylcholine and sodium nitroprusside. CONCLUSIONS AND IMPLICATIONS: NO is the active component of nitroparacetamol in this model of endotoxaemia. Pro-inflammatory processes targeted by nitroparacetamol have been shown to include iNOS/COX-2 induction and possibly vascular soluble guanylyl cyclase. Precise mechanisms underlying the NO effect are unclear but inhibition of cytokine formation may be important.

Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide?

BACKGROUND AND PURPOSE: The aim of these experiments was to evaluate the significance of the chemical reaction between hydrogen sulphide (H2S) and nitric oxide (NO) for the control of vascular tone. EXPERIMENTAL APPROACH: The effect of sodium hydrosulphide (NaHS; H2S donor) and a range of NO donors, such as sodium nitroprusside (SNP), either alone or together, was determined using phenylephrine (PE)-precontracted rat aortic rings and on the blood pressure of anaesthetised rats. KEY RESULTS: Mixing NaHS with NO donors inhibited the vasorelaxant effect of NO both in vitro and in vivo. Low concentrations of NaHS or H2S gas in solution reversed the relaxant effect of acetylcholine (ACh, 400 nM) and histamine (100 microM) but not isoprenaline (400 nM). The effect of NaHS on the ACh response was antagonized by CuSO(4) (200 nM) but was unaffected by glibenclamide (10 microM). In contrast, high concentrations of NaHS (200-1600 microM) relaxed aortic rings directly, an effect reduced by glibenclamide but unaffected by CuSO4. Intravenous infusion of a low concentration of NaHS (10 micromol kg(-1) min(-1)) into the anaesthetized rat significantly increased mean arterial blood pressure. L-NAME (25 mg kg(-1), i.v.) pretreatment reduced this effect. CONCLUSIONS AND IMPLICATIONS: These results suggest that H2S and NO react together to form a molecule (possibly a nitrosothiol) which exhibits little or no vasorelaxant activity either in vitro or in vivo. We propose that a crucial, and hitherto unappreciated, role of H2S in the vascular system is the regulation of the availability of NO.

Prostaglandin metabolism in rabbit kidney. Identification and properties of a novel prostaglandin 9-hydroxydehydrogenase.

Prostaglandin F2alpha is metabolized in 100 000 X g supernatants of rabbit kidney by a 15-hydroxydehydrogenase and a delta13-reductase to F-series metabolites, and byt a 9-hydroxydehydrogenase to E-series compounds. The reactions were measured by radiochemical and biological assays. The 15-hydroxydehydrogenase and 9-hydroxydehydrogenase are localised specifically in renal cortex, have optimal activity at alkaline ph and are effective over a wide range of substrate concentrations. The 9-hydroxydehydrogenase oxidises the 9-alpha hydroxyl group of prostaglandin F2alpha and F1alpha but not the 9-beta hydroxyl group of prostaglandin F2beta. This enzyme is also found in rabbit stomach and ileum, but not in 8 other organs examined; the 15-hydroxydehydrogenase has a wider distribution. Unlike other 9-hydroxydehydrogenase enzymes described to date, the enzyme prepared from rabbit kidney converts prostaglandin F2alpha directly to E2 in substantial amounts; this conversion may be of importance in renal homeostasis.

Selective inhibitors of neuronal nitric oxide synthase--is no NOS really good NOS for the nervous system?

It is now ten years since NO was shown to account for the biological activity of endothelium-derived relaxing factor (EDRF). It is also the tenth anniversary of the identification of L-NG monomethyl arginine (L-NMMA) as the very first inhibitor of NO biosynthesis. That EDRF and NO were one and the same sparked an explosion of interest in the biochemistry and pharmacology of NO which has yet to subside. In contrast, the first ever nitric oxide synthase (NOS) inhibitor slipped seamlessly into the literature virtually without comment at the time. Over the following decade, L-NMMA (and like NOS inhibitors) have proved invaluable as tools for probing the biological roles of NO in health and disease and, in particular, have increased our understanding of the function of NO in the nervous system. Further advances in this important area now require the development of inhibitors selective for the neuronal isoform of NOS (nNOS). Here, Philip Moore and Rachel Handy provide an up-to-date account of the literature regarding the biochemical and pharmacological characterization of NOS inhibitors with particular reference to compounds with greater selectivity for the nNOS isoform.

A unique role for p53 in the regulation of M2 macrophage polarization.

P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological 'brake' to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.

Inhibition of nitric oxide synthase--potential for a novel class of therapeutic agent?

The free-radical gas nitric oxide (NO) plays an important role in a wide and diverse range of physiological processes. As progress is made in understanding the biological function of NO, there is growing interest in the possibility that inhibitors of NO synthase (NOS) may be of clinical use in the therapy of certain disease states. The search for novel and clinically relevant inhibitors of this enzyme represents a truly multidisciplinary approach to drug screening and will no doubt benefit from the application of recombinant DNA (rDNA) technology.

Effects of selective inhibitors of neuronal nitric oxide synthase on carrageenan-induced mechanical and thermal hyperalgesia.

The effect of inhibition of nitric oxide synthase (NOS) on hindpaw hyperalgesia (assessed using mechanical and thermal noxious stimuli) and oedema formation following intraplantar injection of carrageenan (150 microl, 2% w v(-1)) in the rat was determined. For this purpose, NOS inhibitors including L-N(G) nitro-arginine methyl ester (L-NAME; isoform non-selective NOS inhibitor), 7-nitroindazole (7-NI) and 1-(2-trifluoromethylphenyl) imidazole (TRIM; both relatively selective inhibitors of neuronal NOS) were used. Mechanical/thermal nociceptive threshold values and hindpaw weight were recorded prior to and 3 h after administration of carrageenan. NOS inhibitors (5-25 mg kg(-1), i.p.) were administered 2.5 h after carrageenan. L-NAME, 7-NI and TRIM inhibited carrageenan-induced mechanical and thermal hyperalgesia. Calculated ED50 values (micromol kg(-1), i.p.) were 63.4, 96.2 and 92.7 (mechanical) and 42.2, 53.9 and 62.1 (thermal), respectively. None of the drugs affected pain perception in the non-injected hindpaw or carrageenan-induced hindpaw weight gain. Thus, 7-NI and TRIM, at doses previously reported not to influence cardiovascular haemodynamics, inhibit hyperalgesia in the rat regardless of the type of noxious stimulus employed. Accordingly, selective inhibitors of neuronal NOS may prove useful for the treatment of prolonged pain in man.

Endothelium-derived relaxing factor and the effects of acetylcholine and histamine on resistance blood vessels.

1. The role of endothelium-derived relaxing factor (EDRF) in the action of vasodilator (acetylcholine, histamine, nitroprusside) and vasoconstrictor (noradrenaline, vasopressin) drugs on vascular resistance in the isolated perfused kidney and mesentery of the rat was studied. 2. Acetylcholine (EC50 = 0.18 +/- 0.05 nmol and 3.1 +/- 0.06 nmol, n = 8) and histamine (EC50 = 31.2 +/- 4.9 nmol and 46.2 +/- 3.9 nmol, n = 8) produced dose-related vasodilatation in noradrenaline-preconstricted (i.e. 'high tone') rat renal and mesenteric blood vessels. The response to both vasodilators (but not nitroprusside) was abolished by infusion of CHAPS (4.7 mg ml-1, 30 s). By use of an immunocytochemical staining procedure CHAPS was demonstrated to remove vascular endothelial cells lining intrarenal blood vessels. 3. Gossypol (3 microM), metyrapone (10 microM) and nordihydroguaiaretic acid, (NDGA, 30 microM), presumed inhibitors of EDRF biosynthesis, reduced or abolished the response to acetylcholine and histamine in perfused kidney and mesentery of the rat without affecting vasodilatation due to nitroprusside. Mepacrine (10 microM) similarly abolished the response to acetylcholine and histamine but in addition, reduced the response to nitroprusside in both preparations. 4. Methylene blue (100 microM), a presumed antagonist of the effect of EDRF, abolished vasodilatation due to acetylcholine and histamine and reduced the response to nitroprusside in perfused rat kidney and mesentery. Superoxide dismutase, SOD (15 u ml-1), was without effect. 5. While CHAPS treatment significantly augmented the vasoconstrictor response to both noradrenaline and vasopressin in perfused renal and mesenteric vessels this effect was not mimicked by metyrapone or gossypol suggesting that the enhanced effect of vasopressor agents in CHAPSperfused rat organs is due to the removal of a permeability barrier rather than impaired EDRF formation. 6. Responses to vasoconstrictor and vasodilator drugs in the perfused kidney and mesentery were obtained in the presence of indomethacin (8 microM) which produced in excess of 90% inhibition of prostacyclin (PGI2) release as measured by radioimmunoassay of 6-oxo-prostaglandin F1 alpha,. (6-oxo- PGF1 alpha) in the Krebs effluent. 7. We provide evidence that EDRF mediates the vasodilator response to acetylcholine and histamine in resistance blood vessels in perfused rat kidney and mesentery. The possibility that EDRF has a physiological role to play in regulating the calibre of resistance blood vessels is discussed.

Prostaglandins release plasma 'reciprocal coupling factor' in anaesthetized rats.

1. Prostaglandins E1, E2, I2 and endoperoxide analogue U46619 injected intra-arterially (i.a.) into anaesthetized rats at 2 micrograms/kg caused a substantial increase within 60 min of the plasma activity of prostaglandin 'reciprocal coupling factor' (RCF). RCF is the provisional name for the component(s) of plasma which inhibit microsomal prostaglandin synthesis and enhance cytosolic prostaglandin breakdown. 2. RCF is not released by inactive metabolite 13,14-dihydro-15-keto prostaglandin E2 (10 micrograms/kg, i.a.) or acetylcholine or histamine (2 micrograms/kg, i.a.). 3. We suggest that release by prostaglandins of RCF would provide the basis in vivo for a negative feedback mechanism controlling the activity of the prostaglandin system.

The effect of arginine and nitric oxide on resistance blood vessels of the perfused rat kidney.

1. The vasodilator effects of arginine, nitric oxide (NO), acetylcholine (ACh) and sodium nitroprusside (NP) in the noradrenaline-preconstricted ('high tone') perfused rat kidney have been examined. 2. L-Arginine (0.6-23 mumol) caused a biphasic change in renal perfusion pressure. D-Arginine (0.6-23 mumol) was without effect. The second vasodilator component was abolished and the first vasoconstrictor effect augmented following CHAPS-induced removal of the vascular endothelium suggesting that vasodilatation was endothelium-dependent. 3. L-Arginine salts produced transient and dose-related vasodilatation. L-Arginine methylester was the most potent with an ED50 of 2.2 +/- 0.4 mumol (n = 6). The rank order of potency of the salts tested was: methylester greater than hydroxamate greater than chloride. L-Homoarginine chloride was also vasodilator (ED50, 12.0 +/- 1.3 mumol, n = 5). D-Arginine chloride was without effect at doses up to 170 mumol. Responses to L-arginine chloride were endothelium-derived relaxing factor (EDRF)-dependent being abolished by CHAPS (4.7 mg ml-1, 30 s) and significantly inhibited (greater than 70%) by gossypol (3 microM) and nordihydroguaiaretic acid (NDGA, 10 microM). 4. Vasodilatation due to NO was unaffected by CHAPS and gossypol treatment but inhibited by NDGA. NO was approximately 3 times less potent than ACh but 3000 times more potent than L-arginine methylester. 5. Kidneys perfused for 1 h with Krebs solution containing L-arginine chloride (100 microM) or L-canavanine (50 microM) showed no change in sensitivity towards ACh or NP. Higher concentrations of L-arginine chloride (500 microM) or L-canavanine (150 microM) significantly reduced the response to both vasodilators 6. L-Arginine salts dilate resistance blood vessels of the perfused rat kidney by a mechanism which may involve the release of EDRF from vascular endothelial cells of the perfused rat kidney..

A comparison of the effects of L-NAME, 7-NI and L-NIL on carrageenan-induced hindpaw oedema and NOS activity.

1. Intraplantar injection of carrageenan (150 microl, 1-3% w/v) in the rat resulted in a dose-related increase in hindpaw weight (oedema) characterized by a rapid 'early' phase (up to 2.5 h) response followed by a more sustained 'late' phase (2-6 h) response. No change in weight of either the contralateral (i.e. noninjected) hindpaw or hindpaws injected with saline was observed. 2. Six hours after intraplantar injection of carrageenan (1-3% w/v) hindpaw constitutive (i.e. calcium-dependent) nitric oxide synthase (cNOS) activity (determined ex vivo as the conversion of radiolabelled L-arginine to radiolabelled citrulline) was increased (e.g. 2% w/v; 0.64+/-0.08 pmol citrulline mg(-1) protein 15 min(-1) c.f. 0.08+/-0.04 pmol citrulline mg(-1) protein 15 min(-1) in saline-injected, control animals, n=4, P<0.05). Carrageenan injection also resulted in the appearance in hindpaw homogenates of inducible (i.e. calcium-independent) nitric oxide synthase (iNOS, e.g. 2% w/v; 0.67+/-0.14 pmol citrulline mg(-1) protein 15 min(-1), n=4). Hindpaw cyclic GMP concentration was also significantly increased 6 h after intraplantar injection of carrageenan (e.g. 2% w/v; 379.6+/-26.8 fmol mg(-1) protein c.f. 261.8+/-42.2 fmol mg(-1) protein, in saline-injected, control animals, n=4, P<0.05). 3. Pretreatment (5-25 mg kg(-1), i.p., 30 min before carrageenan, 2% w/v) of animals with L-N(G) nitro arginine methyl ester (L-NAME; isoform nonselective inhibitor of NOS) or 7-nitro indazole (7-NI; inhibitor of neuronal NOS, nNOS) caused dose-related inhibition of both the early (2 h) and late (6 h) phase hindpaw oedema, associated with reduced hindpaw iNOS and cNOS activity and cyclic GMP concentration in animals killed at 6 h. Administration of 7-NI (5-25 mg kg(-1), i.p.) to animals 2.5 h after intraplantar carrageenan (2% w/v) injection (i.e. at the end of the early phase oedema response) produced dose-related inhibition of the late phase response. 4. Pretreatment (5-25 mg kg(-1), i.p., 30 min before carrageenan, 2% w/v) of animals with L-N6-iminoethyllysine (L-NIL, selective inhibitor of iNOS) (5-25 mg kg(-1)) failed to affect the early phase hindpaw oedema response but did produce a dose-related inhibition of the late phase oedema. L-NIL pretreatment also inhibited the carrageenan-induced increase in both hindpaw iNOS and cNOS activity as well as the rise in hindpaw cyclic GMP concentration. 5. The present experiments demonstrate an anti-inflammatory effect of 7-NI as evidenced by inhibition of carrageenan-induced hindpaw oedema in the rat. Inhibition of nNOS (early phase) and iNOS (late phase) at the site of inflammation most probably accounts for the anti-inflammatory activity observed. These data suggest a role for nitric oxide synthesized by the nNOS isoform (most probably within sensory nerves) in this model of inflammation.

Effects of sulphasalazine and its metabolites on prostaglandin synthesis, inactivation and actions on smooth muscle.

1 We have investigated the effects of sulphasalazine and of its principal colonic metabolites (5-aminosalicylic acid and sulphapyridine) on prostaglandin inactivation, synthesis and actions on gastrointestinal smooth muscle.2 Sulphasalazine inhibits prostaglandin F(2alpha) breakdown in 100,000 g supernatants in all organs so far tested from 7 species with an ID(50) of approx. 50 muM; it has a selective action on prostaglandin 15-hydroxydehydrogenase and does not inhibit prostaglandin Delta-13 reductase, prostaglandin 9-hydroxydehydrogenase or ;enzyme X' at millimolar concentrations. Enzyme activities were measured radiochemically or by bioassay.3 Sulphapyridine and 5-aminosalicylic acid do not inhibit prostaglandin inactivation in vitro (4 species tested). A methyl analogue of sulphasalazine is a more potent inhibitor than the parent compound. Rabbit colon prostaglandin F(2alpha) metabolism in vitro was inhibited by the following drugs with ID(50) values (muM) of: diphloretin phosphate 20, sulphasalazine 50, indomethacin 220, frusemide 1000 and aspirin 10,000. A similar rank order of potencies was obtained with rabbit kidney.4 Sulphasalazine at 50 to 100 muM inhibited inactivation of prostaglandin E(2) in the perfused rat and guinea-pig lung by 3 to 40% (rat) and 32 to 100% (guinea-pig) when measured by superfusion cascade bioassay and of prostaglandin F(2alpha) by 43.6 +/- 6.5% in rat lung perfused with 50 muM sulphasalazine and assayed radiochemically.5 Prostaglandins E(1) and E(2) were 97.0 +/- 8.2% and 92.3 +/- 6.8% inactivated in the lungs after intravenous injection in the anaesthetized rat as measured by reference to their vasodepressor potencies when injected intra-arterially. Prostaglandin A(2) was not similarly inactivated. Pulmonary inactivation was prevented in the presence of an intravenous infusion of 16.3 mug kg(-1) min(-1) sulphasalazine and partially inhibited at a lower infusion rate.6 Prostaglandin biosynthesis from arachidonic acid was measured in microsomal preparations from four sources by bioassay and radiochemical methods. Indomethacin was a potent inhibitor (ID(50) 0.8 to 4.1 muM) but sulphasalazine and its methyl analogue were very weak inhibitors (ID(50) 1500 to > 5000 muM), 5-aminosalicylic acid was weaker still and sulphapyridine inactive.7 Sulphasalazine at 50 muM did not affect the actions of prostaglandins on five smooth muscle preparations; at 500 muM there was a rapidly reversible and probably non-specific antagonism of responses to low doses of prostaglandins.8 The specificity and selectivity of the interaction of sulphasalazine and its metabolites with the formation, breakdown and actions of prostaglandins are discussed.

Formation of 6-keto prostaglandin E1 in mammalian kidneys.

1 The metabolism of prostacyclin (PGI2) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) was studied in cell-free homogenates of rat, rabbit and guinea-pig kidney. 2 Rabbit kidney converted both PGI2 and 6-keto PGF1 alpha to a stable metabolite with chromatographic and biological activity identical to that of authentic 6-keto PGE1. Activity was found in the kidney cortex but not medulla, was inhibited by NAD+ or NADP+ (5 mM) and showed an optimum temperature requirement of 37 degrees C. 3 Guinea-pig kidney converted PGI2 but not 6-keto PGF1 alpha to a labile, biologically active metabolite which was not 6-keto pge1. 4 No conversion of prostacyclin or 6-keto PGF1 alpha to biologically active metabolites occurred in cell-free homogenates of rat kidney, liver and colon or guinea-pig liver and colon. 5 6-keto PGE1 rapidly lost spasmogenic activity on the rat stomach strip following incubation with rabbit or guinea-pig kidney supernatant in the absence of added cofactors. No loss of activity occurred on incubation with rat kidney. 6 Rutin (50 microM) potently inhibited synthesis of 6-keto PGE1 from added PGI2 by rabbit kidney cortex. This reaction was potentiated by a similar concentration of sulphasalazine, carbenoxolone, imidazole, papaverine or indomethacin. 7 The relevance of these findings for the possible physiological and pathological roles of 6-keto PGE1 in the kidney is discussed.

Conversion of prostacyclin to 6 oxo prostaglandin E1 by rat, rabbit, guinea-pig and human platelets.

The enzymatic catabolism of prostacyclin (PGI2) to 6 oxo prostaglandin E1 (6 oxo PGE1) was studied in platelet-rich and platelet-poor-plasma of rat, rabbit, guinea-pig and man. Rat, rabbit and human platelets convert PGI2 to a product with biological activity and thin layer chromatographic mobility identical to that of authentic 6 oxo PGE1. Platelets from these species also converted 9 beta-[3H]-PGI2 to non-radioactive 6 oxo PGE1 as shown by the progressive loss of extracted radioactivity following incubation. Formation of 6 oxo PGE1 was inhibited by the flavonoid drugs, rutin and naringenin. Guinea-pig platelets did not convert PGI2 to 6 oxo PGE1. Rat, rabbit and guinea-pig platelets do not spontaneously release a 6 oxo PGE1-like substance when incubated at 37 degrees C in the absence of added PGI2 or aggregating agents. The relevance of these findings to the possible physiological and pathophysiological roles of 6 oxo PGE1 in the regulation of platelet function is discussed.

Pathways of prostaglandin F2alpha metabolism in mammalian kidneys.

1 High-speed cytoplasmic supernatants of rat, rabbit, pig and guinea-pig kidneys were prepared and the metabolism of 10 mug/ml prostaglandin F(2alpha) labelled with [(3)H(1)-9beta]-prostaglandin F(2alpha) studied by thin layer radiochromatography and bioassay.2 The metabolism of prostaglandin F(2alpha) measured by radiochromatography parallels biological inactivation in all species except the rabbit.3 Kidneys metabolize prostaglandin F(2alpha) by two divergent pathways, yielding a mixture of prostaglandin E and F metabolites.4 15-Hydroxyprostaglandin dehydrogenase and prostaglandin Delta-13 reductase are present in all species in characteristic proportions. Thus prostaglandin F(2alpha) is metabolized sequentially to 15-keto prostaglandin F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha). The rate and profile of formation of these metabolites is species-dependent.5 13,14-Dihydro-15-keto prostaglandin F(2alpha) is the principal prostaglandin F series metabolite in all species.6 Pig and guinea-pig kidney contain an unidentified enzyme which converts 13,14-dihydro-15-keto prostaglandin F(2alpha) to 13,14-dihydro prostaglandin F(2alpha).7 Rat kidney contains a high concentration of a prostaglandin 9-hydroxy dehydrogenase which converts 13,14-dihydro-15-keto prostaglandin F(2alpha) to 13,14-dihydro-15-keto prostaglandin E(2).8 Rabbit kidney contains a novel 9-hydroxydehydrogenase which oxidises prostaglandin F(2alpha) directly to E(2), thus producing a compound with more potent renal actions. The possible implications of this enzyme for kidney homeostasis are discussed.

Sulphasalazine is a potent inhibitor of prostaglandin 15-hydroxydehydrogenase: possible basis for therapeutic action in ulcerative colitis.

Sulphasalazine is a potent and selective inhibitor in vitro of prostaglandin 15-hydroxydehydrogenase in rabbit colon (ID50 = 50 micrometer) and in several other organs of different species, but does not inhibit prostaglandin delta-13 reductase or microsomal prostaglandin synthesis from arachidonic acid. It is suggested that this action may underly the therapeutic usefulness of sulphasalazine in ulcerative colitis for the prevention of relapse.